Feather-lysate, a hydrolyzed feather feed ingredient and animal feeds containing the same

ABSTRACT

A hydrolyzed feather product, feather-lysate, is disclosed. The product comprises partially hydrolyzed feather, proteins cleaved from the partially hydrolyzed feather, peptides cleaved from the partially hydrolyzed feather, and Bacillus licheniformis cells. Preferably, the B. licheniformis cells are killed. Feather-lysate may be combined with a carbohydrate source to form an animal feed.

FIELD OF THE INVENTION

This invention relates to animal feed ingredients generally, andparticularly relates to a hydrolyzed feather feed ingredient,feather-lysate, useful as a source of dietary protein.

BACKGROUND OF THE INVENTION

Feathers are produced in large quantities by the poultry industry. Thesefeathers provide an inexpensive source of raw material for a variety ofpotential uses. Among other things, there has been considerable interestin developing methods of degrading feathers so they can be used as aninexpensive source of amino acids and digestible protein in animal feed.

Processes for converting feather into animal feed which have beendeveloped to date include both steam hydrolysis processes and combinedsteam hydrolysis and enzymatic processes. See, e.g., Papadopoulos, M.C., Animal Feed Science and Technology 16, 151 (1986); Papadopoulos, M.C. et al., Poultry Science 64, 1729 (1985); Alderibigde, A. O. and D.Church, J. Anim. Sci., 1198 (1983); Thomas and Beeson, J. Anim. Sci. 45,819 (1977); Morris and Balloun, Poultry Sci. 52, 858 (1973); Moran etal., Poultry Sci. 46, 456 (1967); Davis et al., Processing of poultryby-products and their utilization in feeds. Part I. USDA Util. Res. Rep.no. 3, Washington, DC (1961). Disadvantages of these procedures, such asthe degradation of heat sensitive amino acids by steam processes and therelatively low digestibility of the resulting products, have lead tocontinued interest in economical new feather degradation procedureswhich do not require a harsh steam treatment.

Accordingly, an object of the present invention is to provide a processfor hydrolyzing keratinaceous material which does not depend upon steamhydrolysis.

An additional object is to provide a process for convertingkeratinaceous material into amino acids at high yields of the aminoacids.

A further object of this invention is to provide a hydrolyzed featherproduct useful as a feed ingredient which is highly digestible andprovides a good quality source of dietary protein and amino acids.

A still further object of the present invention is to provide aneconomical animal feed which employs a hydrolyzed feather product as adietary amino acid source.

The foregoing and other objects and aspects of the present invention areexplained in detail in the Summary, Detailed Description, and Examplessections below.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a method of degradingkeratinaceous material. The method comprises the steps of combiningkeratinaceous material with Bacillus licheniformis to form afermentation media, and then fermenting the media for a time sufficientto degrade the material. In addition to degrading keratinaceousmaterial, the method of the present invention can be used to produceamino acids. In this case, a fermentation media produced as describedabove is fermented for a time sufficient to produce free amino acidstherefrom, and the free amino acids are recovered from the media.

The method of the present invention can also be used to produce ahydrolyzed feather product. In this latter case, a fermentation mediaproduced as described above, with feather as the keratinaceous material,is fermented for a time sufficient to increase the digestibility of themedia (e.g., by enriching the concentration of digestible proteins andpeptides therein). Preferably, the bacteria in the media are then killedto form a hydrolyzed feather product useful as a feed ingredient (i.e,the media with the bacteria are treated to kill the bacteria).

When the method of the present invention is used to produce free aminoacids or a hydrolyzed feather product, the fermentation step ispreferably an anaerobic fermentation step. Under anaerobic conditions,B. licheniformis does not actively grow. Hence, the utilization of aminoacids by B. licheniformis is decreased and free amino acid production,or enrichment, is increased. In addition, prior to the step of combiningthe feathers with B. licheniformis, B. licheniformis is preferably firstgrown under aerobic conditions (preferably in a liquid culture) toobtain enriched quantities of active bacteria. These procedures, asexplained in detail below, provide an efficient and cost-effective wayto degrade keratinaceous material and to utilize feather.

A second aspect of the present invention is a pure culture of thekeratinaceous material-degrading microorganism having the identifyingcharacteristics of Bacillus licheniformis PWD-1, ATCC No. 53757. B.licheniformis PWD-1 is the preferred microorganism for carrying out themethods described above.

A third aspect of the present invention is a hydrolyzed feather product,which may be produced by the method described above. This productcomprises partially hydrolyzed feather, proteins cleaved from thepartially hydrolyzed feather, peptides cleaved from the partiallyhydrolyzed feather, and B. licheniformis cells (preferably killed B.licheniformis cells). This hydrolyzed feather product may be combinedwith a carbohydrate source and, preferably, minerals and vitamins, toform an animal feed. A second, supplementary protein source mayoptionally be included in the feed.

Additional aspects of the present invention are discussed below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention can be carried out with all types of keratinaceousmaterial, including hair, hooves, and feather. Feather is preferred. Anytype of feather may be employed, including chicken, turkey, and duckfeather. Chicken feather is preferred, and is the material recited inthe text which follows. However, the teaching of this text is applicableto the degradation and utilization of all keratinaceous materials.

Prior to combining feather with B. licheniformis to form a fermentationmedium, the feather should be sterilized to kill bacteria naturallypresent on the feather which might otherwise disrupt the fermentationprocess. This sterilization step may be carried out by any means,including fumigation by contacting feather to formalin or ethylene oxidegas, by contacting feather to steam under pressure, or by combinationsof the same. We have found that, by contacting feather to steam underpressure, even for a time insufficient to sterilize the feather,subsequent bacterial degradation of the feather is substantiallyimproved. Accordingly, sterilization steps in the present inventionpreferably include a step of contacting the feather to steam for a timeand at a temperature sufficient to facilitate the subsequent bacterialhydrolysis thereof, even if this steam treatment step does notaccomplish a complete sterilization of the feather. We have found thatcontacting feather to steam under pressure, in an enclosed chamber, at125 degrees Centigrade for a time as brief as 2 minutes is sufficient tosubstantially enhance the subsequent fermentation hydrolysis thereof. Ifsterilization of feather is carried out solely by steam, the feathershould be contacted to steam under pressure at 125 degrees Centigradefor at least 15 minutes (a 15-20 minute treatment at this temperaturebeing, by definition, an autoclaving step). The time and temperature ofsteam treatment should be less than those employed in commercial steamhydrolysis processes, which employ treatment times of 35 minutes or moreat steam pressures of about 35 p.s.i. or more.

Prior to fermentaton, B. licheniformis can be grown aerobically in anysuitable liquid growth medium (preferably a feather-based liquid growthmedium). The bacteria is preferably grown at a temperature of from about45 to about 55 degrees centigrade. After a sufficient quantity of thebacteria is grown (preferably about 10⁸ colony forming units permilliliter), the liquid growth medium is combined with feather in anysuitable fermentation vessel. Preferably, the liquid growth medium andfeather are combined at a high proportion of feather, so that thefermentation is a semi-solid fermentation. A preferred fermentationmedia comprises, by weight, at least about 1 part of dry feather per 4parts of liquid growth medium, or more preferably about 1 part of dryfeather per 2 parts of liquid growth medium.

Carbohydrate sources used to produce an animal feed according to thepresent invention include, for example, corn, oats, barley, sorghum, orcombinations of the same. These grains are preferably ground into a mealfor use in the animal feed. Supplementary protein sources include, forexample, soy meal, fish meal, blood meal, poultry by-product (groundpoultry offal), meat meal, and combinations of the same. An animal feedis comprised of from about 13% to about 25% by weight of protein fromall protein sources (both hydrolyzed feather and supplemental). Thehydrolyzed feather product may be the sole protein source, but ispreferably from about 2% to about 15% by weight of the feed. Othernutrients in small amounts, such as vitamins, minerals, antibiotics, andother substances or compounds may be included in the feed as required.

Bacillus licheniformis strain PWD-1 was deposited with the American TypeCulture Collection in accordance with the Budapest Treaty on Mar. 23,1988, and has been assigned ATCC Accession No. 53757.

PWD-1 has been found to be a gram positive (but gram variable) bacteria.It is a straight rod-shaped bacteria, the rods being from about 2.1 toabout 3.0 microns long and from about 0.5 to about 1.0 microns wide,with the ends of the rods being rounded. Bacterial cells are found bothsingly and in chains. One subterminal endospore is formed per cell, theendospore being centrally located and cylindrical or oval in shape.

PWD-1 forms opaque, entire (mucoid) colonies which are erose andirregular in shape. Low convex, high convex (mucoid) and flat coloniesare seen. Colonies are seen to disassociate. The colonies are observedas glistening (mucoid) dull, dry, smooth (mucoid) and rough, with aninsoluable brown pigment present. Cells are motile (flagella beingpresent) and peritrichous.

PWD-1 grows at temperatures of from about 20° C. to about 55° C., withtrace growth being present at 60° C. In our hands, the bacteria isthermophilic, growing best at temperatures of from 45° to 50° C. Othershave reported an optimum growth range of between 21 and 30 degreesCentigrade. Experiments are under way to examine the effects ofdifferent growth media on optimum growth temperature.

PWD-1 produces acid, but not gas, from L-arabinose, D-xylose (weakly),D-glucose, lactose (weakly), sucrose, and D-mannitol. It can utilizeboth citrate and propionate as a carbon source. PWD-1 hydrolyzespolysaccharide, starch and casein, but not hippurate. PWD-1 liquifiesgelatin. It reduces, but does not reoxidize, methylene blue. It reducesnitrate to nitrite, but it does not reduce nitrite.

PWD-1 is Voges Proskauer (5198) positive, Voges Proskauer (5198 fil)positive, and Voges Proskauer (5331) positive. It decomposes hydrogenperoxide but not tyrosine, is negative for indole, and is positive fordihydroxyacetone. PWD-1 is negative in the Litmus milk acid test,negative in the Litmus milk coaulation test, and negative in the Litmusmilk alkaline test, but is positive in both the litmus milkpeptonization and litmus milk reduction tests.

PWD-1 grows at a pH of 5.7 and at a pH of 6.0. It shows a pH VP5198 of8.0 or more. The optimum pH in nutrient broth is 7.0 to 7.5. It isaerobic and facultative. It does not grow in 0.02% azide. It generatesgas from sealed nitrate and grows in sealed glucose. It is negative forlecithinase.

The present invention is explained further in the following examples.These examples are provided for illustrative purposes only, and are notto be taken as limiting.

EXAMPLE 1 Comparison of Amino Acid Production By Aerobic and AnaerobicFermentations

Bacillus licheniformis PWD-1 was grown in two batches of a sterileaqueous medium containing feather as the sole source of carbon andenergy. Each liter of medium contained 0.5 grams of NH₄ Cl, 0.5 grams ofNaCl, 0.3 grams of K₂ HPO₄, 0.4 grams of KH₂ PO₄, 0.24 grams ofMgCl₂.6H₂ O, 1.0 grams of hammer-milled feather (a coarsely choppedfeather) and 0.1 grams of yeast extract. The media were adjusted to a pHof 7.5. Each culture was incubated at 50 degrees Centigrade, one beinggrown under aerobic conditions and the other being grown under anaerobicconditions. The quantity of amino acids found in the media at day zero(prior to inocculation) and after five days of incubation under bothaerobic and anaerobic conditions, is shown in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        Free amino acid concentrations in the growth                                  medium of feather degrading microorganisms                                    under aerobic and anaerobic conditions.                                                        Day 5                                                        Amino Acid (mg/l)                                                                          Day 0     Aerobic    Anaerobic                                   ______________________________________                                        ASP (N)      0.00      0.00       3.40                                        THR          0.78      0.90       9.00                                        SER          0.83      1.10       7.60                                        GLU (N)      0.00      1.70       11.80                                       GLY          1.61      0.50       6.60                                        ALA          1.35      1.10       14.50                                       CYS          0.11      3.00       10.20                                       VAL          0.36      2.10       17.10                                       MET          0.00      1.90       4.60                                        ILE          0.19      1.70       20.70                                       LEU          0.91      2.10       32.60                                       TYR          0.00      0.00       6.30                                        PHE          0.00      3.30       22.40                                       ORN          0.00      0.70       7.50                                        LYS          0.99      3.10       6.80                                        HIS          0.22      0.00       2.70                                        ARG          1.05      1.00       11.20                                       TOTAL        8.40      24.20      195.00                                      ______________________________________                                    

These data show that the total production of amino acids wasapproximately 800% greater under anaerobic conditions than under aerobicconditions.

EXAMPLE 2 Amino Acid Yields in Semi-Solid Fermentations

PWD-1 was grown in a liquid media like that described in Example 1(except that 10.0 grams of hammer milled feather per liter was usedinstead of 1.0 gram/liter) for 5 days at 50 degrees Centigrade underaerobic conditions to reach 10⁸ CFU per milliliter. An additionalquantity of hammer milled feathers was autoclaved with steam at 125degrees Centigrade (16 p.s.i.) for 15 minutes. The feathers were mixedwith the growth medium at a proportion of 250 grams feather per liter ofgrowth medium in a fermentation vessel to form a fermentation medium,and the fermentation medium was flushed with nitrogen. The fermentationmedium was then incubated anaerobically for five days at 50 degreesCentigrade with periodic agitation. The same procedure was carried outon a separate occasion, except that the fermentation step was carriedout aerobically. These procedures were duplicated for both conditions.The amino acid concentrations in the liquid phase of all thefermentation media were measured on day five of fermentation, these databeing given in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        Total free amino acid concentrations (g/l) in                                 the liquid phase of the semi-solid phase                                      fermentations                                                                 Fermentation    Anaerobic Aerobic                                             ______________________________________                                        1               14.7      1.7                                                 2               12.4      1.9                                                 ______________________________________                                    

Note that, under anaerobic conditions, free amino acids are produced inquantities in excess of 10 grams/liter by semi-solid phasefermentations.

EXAMPLE 3 Production of a Hydrolyzed Feather Meal Food Product

After five days of anaerobic fermentation, a semi-solid fermentationmedium like that described in Example 2 above was autoclaved with steamat 125 degrees Centigrade for 15 minutes, then dried at 60 degreesCentigrade for 48 hours, and then milled through a one millimeter mesh.The resulting product was a brown powder comprised of partiallyhydrolyzed feather, short peptides, amino acids, and killed Bacilluslicheniformis bacterial cells. This product is useful, among otherthings, as a dietary source of protein for growing chickens, as shown inExample 4 below.

EXAMPLE 4 Animal Feeds Incorporating Bacillus Licheniformis HydrolyzedFeather

One hundred and twenty-eight chicks were divided into four equal groups,4×8 in each group. Each group was raised on a different diet: group (a)was raised on corn-soy feed/20% protein; group (b) was raised oncorn-soy feed/15% protein; group (c) was raised on corn-soy feed/15%protein plus 5% unhydrolyzed hammer-milled feather; group (d) was raisedon corn-soy feed/15% protein plus 5% protein from Bacillus licheniformishydrolyzed feather produced in accordance with the procedure describedin Example 3 above. All birds were weighed at three weeks of age and theweights for each group averaged. These averages are shown in Table 3below.

                  TABLE 3                                                         ______________________________________                                        Ability of growing chickens to utilize feather-                               lysate as a dietary protein source.                                                                Mean Body Weight                                                              (grams)                                                  Diet                 M ± SEM                                               ______________________________________                                        Standard corn-soy, 20% protein                                                                     554.7 ± 8.5                                           Standard corn-soy, l5% protein                                                                     503.9 ± 8.3                                           Corn-soy, l5% protein + 5% protein                                                                 458.8 ± 7.l                                           from untreated feathers                                                       Corn-soy, l5% protein + 5% protein                                                                 525.6 ± 8.9                                           from feather-lysate                                                           ______________________________________                                    

These data show that Bacillus licheniformis hydrolyzed feather can beused as an inexpensive source of dietary protein in animal feed.

The foregoing examples are provided to illustrate the present invention,and are not to be taken as restrictive thereof. The scope of theinvention is defined by the following claims, with equivalents of theclaims to be included therein.

That which is claimed is:
 1. A hydrolyzed feather animal feed supplementconsisting essentially of partially hydrolyzed feather, proteins cleavedfrom said partially hydrolyzed feather, peptides cleaved from saidpartially hydrolyzed feather, and Bacillus licheniformis PWD-1 (ATCC No.53757) cells, said feather hydrolyzed to an extent sufficient toincrease the digestability thereof.
 2. A hydrolyzed feather animal feedsupplement as claimed in claim 1, wherein said Bacillus licheniformisPWD-1 cells are killed Bacillus licheniformis PWD-1 cells.
 3. Ahydrolyzed feather animal feed supplement as claimed in claim 1 producedby fermenting said feather with living Bacillus licheniformis PWD-1bacteria.
 4. An animal feed comprising a carbohydrate source and a firstprotein source, said first protein source consisting essentially ofpartially hydrolyzed feather, proteins cleaved from said partiallyhydrolyzed feather, peptides cleaved from said partially hydrolzedfeather, and killed Bacillus licheniformis PWD-1 (ATCC No. 53757) cells,said feather hydrolyzed to an extent sufficient to increase thedigestability thereof.
 5. An animal feed as claimed in claim 4, whereinsaid carbohydrate source is selected from the group consisting of cornmeal, oat meal, barley meal, sorghum meal, and combinations of the same.6. An animal feed as claimed in claim 4, further comprising asupplemental second protein source.
 7. An animal feed as claimed inclaim 6, wherein said supplemental protein source is selected from thegroup consisting of soy meal, fish meal, blood meal, ground poultryoffal, meat meal, and combinations of the same.